Substitutions in the yeast Nrd1 RNA-binding domain have distinct effects on NNS terminator recognition

RNA-binding proteins (RBPs) regulate gene expression through combinatorial interactions with RNAs and proteins. The essential S. cerevisiae nuclear RBP Nrd1 serves as a model to investigate the effects of RBPs on transcription and RNA processing. Nrd1 binding to short, noncoding RNAs elicits termination of their synthesis by RNA polymerase (RNAP) II, promotes polyadenylation by the TRAMP complex, and enables 3'-trimming by the nuclear exosome. How Nrd1 discriminates its substrate RNAs and coordinates these processes is not well understood. Here, we made deletions and substitutions in Nrd1 to assess the contributions of different domains to its function. We confirmed the contribution of known protein interaction domains in NNS terminator recognition and identified a region C-terminal to the RNA-binding domain (RBD) required for efficient termination in vivo. We also characterized three RBD substitutions, two of which were obtained in a screen for spindle pole body assembly defects and one of which increases RNAP II terminator recognition by S. pombe Seb1, a Nrd1 ortholog. All three caused NNS terminator readthrough in vivo and decreased RNA-binding affinity of the RBD in vitro. Surprisingly, the substitution with the most severe in vitro RNA-binding defect, G344D, exhibited readthrough of only a minority of snoRNA terminators affected by the original nrd1-5 substitution (V368G), which had the least severe RNA-binding defect. Transcriptome analysis showed that Nrd1-G344D caused dysregulation of mRNAs largely distinct from those affected by Nrd1-5 and identified a role for Nrd1-dependent termination in phosphate regulation similar to that of Seb1 in S. pombe. We conclude that the G344D and V368G substitutions in the RNA recognition motif distinguish between some of the diverse substrates of Nrd1.